Combination vaccine for enhancing immunity against brucellosis

ABSTRACT

A vaccine comprising a combination of Brucella “A” and “M” outer-polysaccharides (OPSs) and “R” protein antigens for enhancing immunity against brucellosis is disclosed. The OPS may be obtained from different strains or species of Brucellae (i.e. combining OPS extracted from different bacteria expressing “A” or “M” OPS, or combining OPS and OPS-protein complexes extracted from different bacteria). The OPS or OPS-protein complexes may also be obtained from a single strain expressing more than one OPS (e.g. from  B. suis  strain 145 which expresses “A”, “M” and possibly other OPSs). The vaccine according to the present invention overcomes the limitation of previously discovered  B. abortus  “A” OPS which only protects against species and strains of Brucella that had “A” OPS but not against others with different OPS.

The present application claims benefit of U.S. Provisional ApplicationSer. No. 60/170,765, filed Dec. 15, 1999.

FIELD OF INVENTION

The present invention relates to a vaccine, comprising a combination ofbacterial components derived either from different species of Brucellae,or one strain expressing different components, that enhances immunityagainst brucellosis. The vaccine formulations are applicable for one ormore cross-reactive bacteria thereof.

BACKGROUND OF THE INVENTION

Brucellosis is a debilitating disease that can cause abortions andweight loss in animals as well as undulating fevers, night sweats,incapacitation and arthritis in humans. It is very hardy toenvironmental factors, easily aerosolized and infectious through skinabrasions, ingestion and the pulmonary route. It is difficult to treatwith antibiotics and often persists as a life-long infection.Brucellosis is a disease endemic to most countries, especiallyunder-developed nations where Brucella species infect 0.1 to 10% of thelivestock such as cattle, swine, sheep, goats, and camels. A zoonoticdisease, these also infect other domestic animals such as dogs andpoultry, wildlife such as bison, caribou and wolves and marine mammalssuch as whales and dolphins. People are especially vulnerable toinfection either through handling infected products or ingestingcontaminated foods.

Up-to-date, effective treatment against brucellosis for animals,including humans, has been limited. For humans, administering high dosesof combination antibiotics, for example doxycycline with rifampin overlong periods, has been found to be effective to clear the disease, butnon-compliance and relapses are common. For animals, the cost andlimited effectiveness of antibiotic treatments often lead to thedecision of either no treatment or elimination of the infected animaland its associated herd.

The most preferred type of disease management is to avoid infection andto reduce the incidence and spread of the disease by vaccination. Forlivestock, namely cattle, at present vaccination consists of using anattenuated (weakened) vaccine strain such as Brucella abortus strain 19.Although it is one of the best vaccines for cattle, it does havelimitations in that the vaccine does not give absolute protection andthere is about a 20% failure rate, results from serological tests can beconfusing for a positive serology may be caused by vaccination,infection, or vaccination with subsequent infection, the vaccinealthough tolerated by cattle is pathogenic for humans, and on occasionthe vaccine does revert to a “wild” or virulent form.

For humans, there existed a French vaccine that consisted of a phenolinsoluble residue. However, this vaccine has been discontinued as it wasfound that the residue caused a high rate of reactogenicity (in onestudy, a large percentage of the vaccine recipients developed swollenlymph glands and granuloma at the site of injection) andhyper-sensitivity (vaccinates that touched killed Brucella preparationspresented symptoms of anaphylactic shock).

Recently, the Applicant has discovered a new vaccine that protectedanimals (e.g. mice, guinea pigs and swine) from brucellosis and whichmay upon further development be suitable for protecting humans. Thevaccine is as described in U.S. Pat. No. 5,951,987 which is hereinincorporated by reference. The vaccine consists of anouter-polysaccharide (OPS) isolated from Brucella such as Brucellaabortus. The vaccine protected animals from different strains andspecies of Brucella tested (e.g. B. abortus 30, B. abortus 2308 and B.suis biovar 1) as well as infections from Francisella tularensis livevaccine strain (LVS) which causes tularemia in mice. This gave evidencethat the vaccine would likely offer effective protection againstinfections from a broad spectrum of Brucella species and cross-reactivebacteria. However, it has subsequently been found otherwise. Althoughthe B. abortus OPS vaccine was effective in offering animals protectionfrom brucellosis, it did so only against species and strains thatresembled B. abortus in serology (i.e. had “A” OPS antigens). It did notappear to be effective against species and strains that resembled B.melitensis in serology (i.e. had the “M” or “A&M” OPS antigens). Hence,there still remains a need for a vaccine which is effective againstinfections from a wide spectrum of Brucella species.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a vaccine comprising a combination of Brucella “A” and “M”outer-polysaccharides and “R” protein antigens. Theouter-polysaccharides may be obtained from the same or different speciesof Brucellae. The most preferred source of OPS is derived from Brucellabut a logical extension of this finding is to use bacterial speciescross-reactive thereof.

The combination may be obtained from combining “A” outer-polysaccharidesextracted from Brucella species selected from the group consisting of B.abortus biovar 1, B. abortus biovar 2, B. abortus biovar 3, B. abortusbiovar 6, B. melitensis biovar 2, B. suis biovar 1, B. suis biovar 2, B.suis biovar 3, B. neotomae and B. maris; “M” outer-polysaccharideextracted from Brucella species selected from the group consisting of B.abortus biovar 4, B. abortus biovar 5, B. abortus biovar 9, B.melitensis biovar 1, B. suis biovar 5; and “R” core polysaccharide andproteins extracted from Brucella species selected from the groupconsisting of B. ovis and B. canis.

Alternatively, the combination may be obtained by combining “AM”outer-polysaccharides extracted from Brucella species selected from thegroup consisting of B. abortus biovar 7, B. melitensis biovar 3 and B.suis biovar 4 (note: B. suis 145 biovar 4 is used in the present patentsubmission), and “R” core polysaccharide and protein extracted fromBrucella species selected from the group consisting of B. ovis and B.canis.

In accordance with another aspect of the present invention, there isprovided a vaccine comprising a combination of Brucellaouter-polysaccharides containing the “A” and “M” antigens and a Brucellaouter-polysaccharide-protein complex.

In this case, the combination may be obtained by combining “A”outer-polysaccharide purified from Brucella species selected from thegroup consisting of B. abortus biovar 1, B. abortus biovar 2, B. abortusbiovar 3, B. abortus biovar 6, B. melitensis biovar 2, B. suis biovar 1,B. suis biovar 2, B. suis biovar 3, B. neotomae and B. maris; “M”outer-polysaccharide purified from Brucella species selected from thegroup consisting of B. abortus biovar 4, B. abortus biovar 5, B. abortusbiovar 9, B. melitensis biovar 1, B. suis biovar 5; and anouter-polysaccharide-protein complex selected from the group consistingof outer-polysaccharide and Brucella membrane proteins,outer-polysaccharide and Brucella surface proteins, outer-polysaccharideand Brucella surface enzymes and outer-polysaccharide and Brucellacytoplasmic proteins.

Alternatively, the vaccine may be obtained by combining “AM”outer-polysaccharides extracted from Brucella species selected from thegroup consisting of B. abortus biovar 7, B. melitensis biovar 3 and B.suis biovar 4, and an outer-polysaccharide having a protein selectedfrom the group consisting of Brucella membrane proteins, Brucellasurface proteins, Brucella surface enzymes and Brucella cytoplasmicproteins.

The vaccine may consist of 1 ng to 10 ug, preferably 1 ug, of each ofthe OPS forming the combination for vaccination of mice weighing about20 grams.

The vaccine is effective as a prophylactic treatment from infectionagainst a wide range of Brucella species namely B. abortus, B.melitensis and B. suis. By logical extension the vaccine is likely to beeffective for the prevention of brucellosis from B. ovis, B. canis, B.neotomae and B. maris. Animal studies support its use as a vaccine forlivestock, and with further development possibly as a vaccine forhumans. It is most effective by intra-peritoneal, sub-cutaneous andintramuscular administration. It is least effective when givenintra-nasally. The vaccine works best against the most virulent speciesand strains of Brucella, most of the healthy vaccinates having nobacteria in their spleens or having a million fold less bacteria thancontrols. The vaccine works, but is less effective where it is notneeded, or in mice given Brucella species and strains of low virulence.

Serum or white blood cells of mammals vaccinated with the vaccine inaccordance with the present invention prevented brucellosis in recipientanimals. Protection is long term (i.e. at least several weeks) butunlike other vaccines it is also protective in the short term (i.e.protective in 1 day or less).

DETAILED DESCRIPTION OF THE INVENTION

Brucella species can be classified by their different type ofouter-polysaccharides (OPS). These serological types are those havingthe “A” OPS, the “M” OPS and those lacking OPS or the “R” group (i.e.antigens are predominantly protein with some antigenicity being the“core” polysaccharides attached to the lipopolysaccharide, or LPS,lacking OPS), wherein “A”, “M” and “R” stands for the type of antigens.Some species also express more than one antigen, for example somestrains of B. suis (biovar 4) which express both the “A” and “M”antigens, and some are capable of changing their antigens within thesame host (personal communications, Dr. G. G. Schurig, 1997). Thevarious types of OPS are very similar in chemical structures. They aregenerally made of identical sugars, which are linked differently.Because of the similarity between the OPS and the considerablecross-reaction between the “A” and “M” OPS of Brucella, one would expecta single OPS vaccine, i.e. a vaccine consisting of one type of OPS, tobe effective against a wide range of Brucella species. This, however,was found to be only partially true. The Applicant discovered that acombination or “cocktail” vaccine, i.e. a vaccine having a combinationof the different OPS, is much more effective than the single OPS vaccinein providing protection against a wide spectrum of Brucella species.

To be most effective, the cocktail or combination vaccine should includea range of OPS, for example both “A” and “M” OPS, and “R” antigens. Asit is likely that one or more OPS, other than “A” and “M”, are alsoproduced (Vizcaino, N., Cloeckaert, A., Zygmunt, M. S., andFermandez-Lago, L. 1999. Molecular characterization of a Brucellaspecies large DNA fragment deleted in Brucella abortus strains: evidencefor a locus involved in the synthesis of a polysaccharide. Infection andImmunity, 67: 2700-2712), the inclusion of these would likely offergreater protection.

The cocktail or combination vaccine may be replaced by anouter-polysaccharide-protein complex. Brucellae can attach proteins (the“R” antigen, which can also be extracted from cells that do not expressthe “A” or “M” antigen, have colonies rough in appearance and expressmainly proteins) to OPS. OPS and OPS-protein complex can be separated(e.g. with 0.2 M trichloroacetic acid, OPS remains soluble, OPS-proteinprecipitates).

Outer-polysaccharides containing the “A” antigen can be obtained from B.abortus biovar 1, B. abortus biovar 2, B. abortus biovar 3, B. abortusbiovar 6, B. melitensis biovar 2, B. suis biovar 1, B. suis biovar 2, B.suis biovar 3, B. neotomae and B. maris.

Outer polysaccharides containing the “M” antigen can be extracted fromB. abortus biovar 4, B. abortus biovar 5, B. abortus biovar 9, B.melitensis biovar 1, B. suis biovar 5.

Proteins, core polysaccharides and short chained outer-polysaccharidescomprising the “R” antigen can be purified from B. ovis and B. canis.

Outer-polysaccharides containing both the “A” and “M” antigens can bepurified from B. abortus biovar 7, B. melitensis biovar 3 and B. suisbiovar 4.

Suitable proteins for the “R” component are Brucella proteins.Preferably, they are outer-membrane proteins (opm) such as opm1, opm2and opm3, lipoprotein linked to cell wall, porin (a protein that allowsions or metabolites through the membrane), A5 on the cell surface,surface proteins such as a, b and X, surface enzymes such as protease,Brucellin proteins and internal or cytoplasmic proteins. Other proteinsmay be mannosyltransferase, GDP-mannose 4,6 dehydratase, perosaminesynthetase, ABC-type transporter and formyl transferase. Additionalproteins such as those identified in the paper “Conservation in Brucellaspp. of seven genes involved in the biosynthesis of thelipopolysaccharide O-chain”, A. Cloeckaert, M. Grayon, J-M Verger, J-JLetesson and F. Godfroidt, 1998. 51th Annual Brucellosis Meeting,Chicago, herein incorporated by reference, can also be used.

To assess the effectiveness of the vaccine, different single OPS vaccineand combination OPS vaccines were prepared and tested. The novelformulation of using combination OPS vaccine of the present invention isshown to be more effective than a single OPS vaccine formerly disclosedby the Applicant in the U.S. Pat. No. 5,951,987.

Materials and Methods

Bacterial Cultures

B. abortus 30, B. abortus 2308, B. melitensis 16M and B. suis 145 wereacquired in 1989 from the Animal Diseases Research Institute(ADRI-Nepean, now the Canadian Food Inspection Agency), Nepean, Ontario,Canada. The bacteria were thawed and small aliquots of the materialswere obtained and grown on Brucella agar (Difco Laboratories, Detroit)with 1.5 ppm crystal violet at 37° C., 5% CO₂, 90% humidity, for 1 week.A loopful (about a billion cells) of the culture were placed in vialscontaining 1 ml of sterile Brucella broth with 15% glycerol and thevials were frozen at −70° C. As required, vials containing B. melitensis16M, B. suis 145, B. abortus 30 and 2308 were thawed and subculturedonto, for example, Brucella agar and incubated to provide cells forProtect Beads™ storage, or into Brucella broth and incubated to providecells which were later used in the infectivity experiments.

Representative vials of the bacteria were thawed and used to inoculateBrucella agar slants (2 cc agar in a 5 cc vial). The cultures wereverified on May 10, 1999 by the National Veterinary ServicesLaboratories (Ames, Iowa), which confirmed that the vials containing B.abortus 30 or B. abortus 2308 belonged to B. abortus biovar 1 (“A”antigen predominant), B. melitensis 16M belonged to B. melitensis biovar1 (“M” antigen predominant), B. suis 145 belonged to the atypical B.suis biovar 4 (has both “A” and “M” antigens).

The bacteria used to cause Brucella infection were prepared byinoculating bacteria such as B. melitensis 16M into Brucella broth(Difco Laboratories, Detroit), grown overnight, then 0.010 ml wastransferred to 100 ml Brucella broth and used within 18 hours ofincubation. This method revealed more effective in providing bacteriawith high virulence than the conventional method of simply thawing afrozen stock of bacteria and determining the colony forming unit (CFU),or adding the thawed stock to prewarmed Brucella broth and incubatingfor 2 hours at 37° C., 5% CO₂ and 90% humidity.

The best way to ensure virulence of the bacterium (e.g. B. melitensis16M) was to passage it through sets of 5 mice. Hence a culture thatappeared to have lost its virulence, would be given to each of 5 mice byintra-peritoneal injection. In this case, each mouse was given 5×10⁴ to5×10⁵ bacteria/0.1 ml sterile saline. After 1 week, the mice weresacrificed, their spleens weighed and crushed in saline, and seriallydiluted and plated on Brucella agar with 1.5 ppm crystal violet andincubated for 2 hours at 37° C., 5% CO₂ and 90% humidity and the colonyforming unit was determined. Bacteria on these plates that were from themouse with the largest spleen size and greatest bacterial number in thespleen was selected and these were used to infect another 5 mice and theselection was repeated. Within 3 passages, the bacterium had exceptionalvirulence (caused large spleens, 3-5 fold larger than normal, and highbacterial numbers, about 3 million bacteria per spleen). These bacteriawere used only for a few experiments as it was viewed that passagethrough an animal could introduce other unknown variables.

Vaccine Preparation

a) Preparation of “A” OPS, “M” OPS or OPS-protein Vaccines

The method of preparation for some of the vaccines (e.g. “A” OPS from B.abortus 1119-3, “M” OPS from B. melitensis 16M) was as previouslydescribed (“Antigens of Brucella”, J. W. Cherwonogrodzky et al., pages54-55, In: K. Nielsen, J. R. Duncan (ed.) 1990 Animal Brucellosis CRCPress, Boca Raton). Briefly, each bacterium was grown on about 90 ml ofBrucella agar with 1.5 ppm crystal violet into each of twenty 150 cm²sterile tissue culture flasks and incubated for 2 hours at 37° C., 5%CO₂ and 90% humidity. After the incubation period, 5 ml of sterile 3%acetic acid with 1% saline was added to each flask, a dozen small glassbeads were added, and the cells made into a suspension by rocking orlightly shaking the flask as required. The suspension was removed with apipette and placed into a 250 ml centrifuge bottle. Another 5 ml ofsterile acetic acid with saline in triply distilled water was added tothe flask. The flask was rocked or shaken as required, and thesuspension was added to the previous one.

Once all twenty flasks were processed, the suspension was shakenvigorously, and stored at 4° C. for at least one week. Thereafter, itwas autoclaved at 121° C., 15 psi for 2 hours with a loosened cap. Onceautoclaved and cooled, the bottle was centrifuged at 15,000×g for 30 minat 4° C. The supernatant was kept and the cells discarded.

The supernatant was neutralized with 10 M NaOH and had 2 Mtrichloroacetic acid (TCA) added to a final concentration of 0.2 M TCA.This was centrifuged (20,000×g, 30 min, 4° C.). The OPS remained solublein the supernatant, the OPS-protein that was precipitated by the TCA wasin the pellet. The pellet was resuspended in triply distilled water.Both the supernatant and the redissolved pellet (kept separate) wereextracted with equal volumes of liquified phenol (90% phenol with10%water) added. The mixture was magnetically stirred for 30 min at 70°C. and was chilled at 4° C. overnight. The bottom phenol layers wereremoved and centrifuged as before to remove debris or the remainingwater layer. To the phenol layers were added 5 volumes of methanol with1% sodium acetate to enhance precipitation and this was chilledovernight at 4° C. The preparations were centrifuged as before, washedtwice more with a similar volume of methanol-acetate, and the pellet wasdissolved in triply distilled water and dialyzed (1000 mw cutoff)against triply distilled water. Once dialyzed, these samples werecentrifuged to remove denatured material and lipopolysaccharide (LPS,this aggregates in distilled water while outer-polysaccharide (OPS)remains soluble). The samples were freeze-dried, weighed and kept at−70° C. until required. The resulting samples were at least 90% pure.

b) Preparation of B. suis 145 OPS Vaccine

When the previous method for OPS preparation was applied to B. suis 145,it was unsuccessful. Neither OPS nor OPS-protein precipitated whenmethanol-acetate was added to the phenol extracts. The material wasstill present, as evidenced by OPS and OPS-protein precipitating whenthe phenol/methanol-acetate solutions were kept at −20° C. instead of 4°C. for a week, but it was clear that the procedure had to be changed toprepare OPS or OPS-protein for vaccines. (It was subsequently found thatB. suis 145 OPS is more heterogeneous in composition than the otherBrucella OPS cited, explaining the failure of the previous method, Dr.Brad Berger, unpublished results.)

In brief, B. suis 145 was grown on agar medium (Brucella agar, with orwithout 1.5 ppm crystal violet, trypticase soy agar, with or without 1.5ppm crystal violet, gave similar results) in sterile 150 cm tissueculture flasks, at 35° C., 5% CO₂ and 90% humidity for 1 week. Afterthis time, cells were both killed and removed by adding 5 ml of 5%phenol/1% saline, adding glass beads, and rolling or shaking the flaskto dislodge the cells, removing the cells, adding another 5 ml ofphenol-saline, rolling and shaking the flask again, and pooling cellsuspensions. From 400 flasks, about 250 grams wet weight of cells wasremoved and the final volume was about 3 liters in sixteen 250 mlcentrifuge bottles. The suspension was kept 1 week at 4° C., withoccasional shaking, to ensure release of loosely bound antigens.

After 1 week, the suspension was centrifuged (15,000×g, 20 min, 4° C.),the supernatants pooled, and the cells washed with a small volume (40 mlper centrifuge bottle, centrifugation as before) of phenol-saline. Theliquid was added to the pooled supernatants.

To the supernatant, glacial acetic acid was added to a final volume of3%. This suspension was then placed in a boiling water bath for 2 hours.It was left to cool to room temperature for a day. The pH was notadjusted (the low pH appears to enhance precipitation at a latermethanol stage). A one-half volume of 90% phenol (10% water) was added(the smaller volume concentrates the OPS). This was magnetically stirredon a magnetic hot-plate until the temperature rose to the mixture's“clarity” point (the phenol-water mixture was opaque, but around 65-70°C. the phenol and water dissolved into each other, causing the mixtureto clear). It was then allowed to cool at 4° C. overnight, centrifugedand the phenol layer (bottom layer, usually dark red in colour) kept. A2-week sterility check is done to ensure this phenol layer is sterileand it is then taken out of Biocontainment 3. Once in the generallaboratory area, the phenol was chilled overnight at 4° C. as was themethanol-acetate (methanol with 1% sodium acetate.3H₂O). Five volumes ofmethanol-acetate was added to the phenol layer, this was mixed on amagnetic stirrer and allowed to settle for 1-2 days. After this time,most of the liquid above the precipitate was aspirated away (the flaskis placed on ice to prevent mixing if it begins warming to roomtemperature). The remaining preparation was centrifuged as before, thesupernatant discarded, and the pellet resuspended in methanol-acetate (asaw-toothed OmniMix™ is best for blending the suspension) andcentrifuged. The pellet was then resuspended in distilled water, placedin a 1000 mw cutoff dialysis cellulose membrane, and dialyzed againstdistilled water at 4° C. (From the frothing that occurred when theoutside water was discarded during changes, it appeared thatconsiderable amount of small m.w. OPS was lost in this process, but thata large amount of larger m.w. OPS was retained by the dialysis.) Afterdialysis, the preparation was removed from the dialysis bag, andcentrifuged to remove denatured material. The pellet of denaturedmaterial was discarded, the supernatant was kept, and to the latter 2 Mtrichloroacetic acid (TCA) was added until the final concentration was0.2 M TCA. This was then centrifuged. Both the supernatant (containingOPS) and the pellet (containing OPS-protein, this was suspended indistilled water) were dialyzed against distilled water. After extensivedialysis, each preparation was centrifuged to remove denatured orparticulate material. The solutions were then aliquoted andfreeze-dried. The OPS could be further refined with enzyme digestion(though the starting material was at least 90% pure, having nodetectable A260/A280 nm absorbing nucleic acids and only about 0.6%protein) and ultra-centrifugation (120,000×g, 4° C., 3 hours) to removetrace amounts of LPS.

For the washed B. suis 145 cells noted previously, these were suspendedin 3% acetic acid and 1% saline (for every gram of cells, 5 ml of aceticacid-saline was added) and placed in a boiling water bath for 2 hourswith swirling to mix the suspension every half hour. The cells were leftfor a day to cool to room temperature. The preparation was centrifuged,and the supernatant kept. The cells were mixed in an equal amount (w/v)of acetic acid saline, centrifuged, and the liquid pooled with theother. A phenol extraction was done (half a volume of phenol was usedwith the cell supernatant) on this liquid as noted before. The OPSreleased from the cell by boiling water was by the method noted above.

The cells from the above were resuspended in 5 volumes of aceticacid-saline, autoclaved (121° C., 15 psi, 2 hours), cooled, centrifuged,and the liquid (as well as a washing of the cells with an equal amountof acetic acid-saline which was added) was processed as before. Thedifferent fractions noted above have been tested (1 ug/0.1 ml sterilesaline/mouse, intraperitoneal injection). All OPS fractions and allOPS-protein fractions were protective (about 10,000 fold less bacteriain their spleens than controls 1 week after challenge). The whole cellwith OPS removed, or the interphase material (between the phenol andwater layers during extraction) were not protective (indeed, theinterphase material was immuno-suppressive, causing mice to have about5-fold more bacteria in their spleens than non-vaccinated control mice).

About 4-fold more OPS was acquired if the B. suis 145, was passagedthrough a mouse before being grown on agar. There was also aconsiderable amount of brown gelatinous material on the cells afterautoclaving (about 20 c.c. on 250 grams of cells, when 2 cc of this wasfreeze-dried and gave 80 mg dry weight). However, as passaging through amouse might be introducing new variables, frozen stocks kept onProtectBeads™ at −70° C. were used as the inoculum for OPS andOPS-protein production.

The strength for using B. suis 145 as a source of vaccine is that as itexpresses both “A” and “M” OPS (as well as Brucella “R” proteins) it isalso likely to express other OPS. Recently another OPS has beenisolated, by a method unobvious to anyone skilled in the art. There wasan insufficient amount of this polysaccharide to characterize, otherthan it was polysaccharide, but animal studies showed that this OPS(which is in the OPS vaccine preparation) also protects mice frombrucellosis (Dr. Brad Berger, unpublished results).

As stated previously in this text, the B. suis 145 OPS or OPS-proteinpreparations were found to be potent vaccines that protected mice frombrucellosis. Against the most virulent strains and species of Brucella,vaccinated mice often had no bacteria in their spleens, or only a few(i.e. a million fold less than controls). For the latter, it was unknownif these were simply the last traces of the challenge that were soon tobe cleared (which is likely for their growth lag on agar medium suggeststhat these are heavily damaged by the immune system of the host) orwhether these were mutants with polysaccharides different from what themice were vaccinated against. These “survivors” were allowed to continuegrowing on the agar plates previously used to assess spleen bacterialloads (35° C., 5% CO₂, 90% humidity, for an additional 3 weeks), thesewere scraped from the plates and suspended (3.3 grams wet weight ofcells was recovered) in 5% phenol, 1% saline (50 ml) and processed asnoted above. Mice have been vaccinated with B. suis 145 OPS (as notedabove, 1 ug/mouse by intraperitoneal injection), or OPS prepared from B.suis 145 bacterial “survivors” from vaccinated mice that were challenged(1 ug/mouse by i.p.), or B suis 145 OPS+B. suis 145 “survivors” OPS(both 1 ug/mouse by i.p.). This study is underway but at the time ofthis patent submission it has not determined if the OPS from bacterial“survivors” is equivalent to B. suis 145 OPS for protecting mice frombrucellosis or whether it acts synergistically to enhance vaccineefficacy.

Mice

Unless otherwise specified, mice were 19-21 grams at the start of theexperiment. They were female balb/c mice obtained from Charles River,St. Contance, Quebec, Canada.

Vaccination

Mice were vaccinated intra-peritoneally (i.p) into the lower right sideof the belly, sub-cutaneously (s.c.) into the nape of skin bunched onthe back, intramuscularly (i.m) into the upper left leg or intra-nasally(i.n.). The intra-nasal route required mice to be anaesthetized withMetofane™. Generally, the vaccines comprised OPS from B. abortus 1119-3,B. melitensis 16M and/or B. suis 145 in sterile saline as a single doseper mouse, or a combination of OPS and OPS-protein. For example, asingle antigen vaccine can be 1 ug of B. suis OPS in 0.1 ml sterinesaline as single dose per mouse. An example of a combination vaccine canbel ug each of B. abortus OPS+1 ug B. melitensis OPS-protein+1 ug B.suis OPS together in 0.1 ml sterile saline as a single dose per mouse.Typically, for intra-peritoneal, sub-cutaneous and intra-muscularinjections, the OPS were diluted in 0.1 ml sterile saline, whereas forintra-nasal installation these were in 0.01 ml (half given into eachnostril). Unless otherwise specified, mice were allowed to rest for 5weeks before challenge.

Oral administration of the vaccine was proven to be effective in thecase of B. abortus OPS vaccine to swine in Venezuela (U.S. Pat. No.5,951,987). While the Applicant did not test oral administration of thenew vaccine formulations of the present invention, it is suggested thatefficacy of these vaccines do extend to oral administration.

Challenges

For intra-peritoneal challenge, a culture of Brucella was dilutedserially in 9 ml sterile saline blanks and 5×10⁵ bacteria (as confirmedby plating) in 0.1 ml of sterile saline was given to each mouse. Themice were allowed to rest for 1 week before being sacrificed andassessed. Past studies suggested that infection by intra-nasalinoculation was more difficult to take place (Cherwonogrodzky J. W., andDi Ninno, V. L. 1994, Brucella, brucellosis, undulant fever, AB—Is it athreat? A review in question and answer form (U), Suffield Memorandum1434, Defence Research Establishment Suffield, UNCLASSIFIED, page 6).Therefore, for intra-nasal challenge, 0.010 ml of a culture broth at anearly phase of growth (about 5×10⁷bacteria/0.010 ml) was used withoutdilution and the mice were allowed to rest for 2 weeks rather than 1week before being sacrificed and assessed.

Assessment of Infection

Mice seldom show any symptoms when infected with Brucella, although withthe more serious strains they may show ruffled, grayish looking fur.Hence the only way to assess infection, was to weigh each mouse,sacrifice these by cervical dislocation and remove organs such asspleens for weighing and obtaining bacterial counts. In this case, thespleens were weighed for the ratio spleen wt/body wt., then crushed in 1ml sterile saline by hand with a glass tissue grinder (Wheaton, 2 mlvolume). This suspension was removed, another 1 ml sterile saline wasadded to the chamber and crushing continued to complete the task and torinse the inside of the chamber. This second 1 ml, was pooled with thefirst. To prevent possible aerosol generation, the work was performedinside a Biosafety 2a or 2b Cabinet inside a Biocontainment Level 3(BL-3) area, and the investigator wore a seam sealed positive pressurehood (3M), HEPA filter with a blower powered by a battery pack, a sealedTyvek overall, double gloves and boots. Five tissue grinders were usedfor each group of mice and these were sterilized between groups. Eachtissue grinder had the chamber filled with 70% ethanol and the grindinghandle inserted therein. The chamber was topped up with 70% ethanol,sprayed with the ethanol to decontaminate the outside and then allowedto sit for 30 minutes. Thereafter, the ethanol was poured out, the topof the chamber and the grinding handle wiped with a KimWipe™ soaked in70% ethanol and the grinding handle allowed to air dry. The ethanol wasremoved from the chamber with a sterile pipette and the chamber rinsedwith sterile saline. Any adhering liquid was removed with anothersterile pipette. For the crushed spleen in 2 ml of saline noted above,0.1 ml of this was plated onto a plate of Brucella agar with 1.5 ppm ofcrystal violet, and 1 ml was transferred to a 9 ml sterile saline blankand dilutions with plating were repeated for these. Plates wereincubated for 2 hours at 37° C., 5% CO₂ and 90% humidity and the CFUcounted after one week incubation.

Separation of Blood Components

In instances where only serum was required, mice were given a doubledose of 1:14 diluted Somnitol™ (pentabarbitol), in 0.5 ml per mouse.Once anesthetized, a heart puncture was done with a 1 ml syringe fittedwith a 26-gauge needle, and whole blood was removed. Mice did notrecover from the amount of anaesthetic given. The blood was transferredto a 1.5 ml Eppendorf™ microcentrifuge tube and the tube was left in therefrigerator at 4° C. for a few hours to clot. It was then vortexedbriefly to loosen the clot and centrifuged at 2000×g for 5 minutes atroom temperature or at 22° C. to separate the serum, which formed thetop layer, from blood cells. The serum was removed with a Pasteurpipette, pooled with serum from other mice in the group, and filteredthrough a 0.2 um filter. It was used within a few hours of preparation.

To fractionate serum into different molecular weight groups, whole serum(about 10 ml from 20 vaccinated mice) was placed initially into a 1000molecular weight (m.w.) cutoff dialysis bag, and dialyzed in a 100 mlgraduated cylinder against distilled water with magnetic stirring withina 4° C. refrigerator. After 24 hours, the dialysate (1000 or less m.w.components), which remained in the cylinder, was frozen andfreeze-dried. The serum was transferred to a 12,000 m.w. cutoff dialysisbag and dialyzed as before. This dialysate was 1,000-12,000 m.w. andfreeze-dried. The serum within the dialysis bag had 12,000 or greaterm.w. components and was freeze-dried.

To separate blood components, initially 1 ml of sterile saline was addedto a 10 ml blood collection tube with heparin (10 fold heparin).One-tenth ml of 10-fold heparin was drawn into the 1 ml syringe used tocollect mouse blood. Whole blood (2 ml) was layered onto an equal volumeof Lymphoprep™ (Accurate Chemical and Scientific Corporation, Westbury,N.Y., USA) and centrifuged 1000×g for 30 minutes at room temperature.The top layer was serum which was drawn off with a Pasteur pipette andthen filtered through a 0.2 μm filter (final volume was about 1.5 ml).Mononuclear and polymorphonuclear cells formed 2 bands within thedextran solution. These were both drawn by a Pasteur pipette and washedtwice with sterile saline (diluted in 0.85% sterile saline) andcentrifuged 2000×g for 30 min at room temperature. The supernatant layerwas discarded and the cell pellet resuspended in 5 ml sterile saline,and washed as before and the cell pellet was resuspended in 1 ml sterilesaline. The red blood cell pellet was removed, washed with sterilesaline and resuspended in 1.5 ml of sterile saline.

The averages and standard error about the mean were calculated using theGraphPad Instat program (version 1.14).

RESULTS AND DISCUSSION EXAMPLE 1 Cross-protection Study

In this study, female balb/c mice were injected intra-peritoneally(i.p.) with either 0.1 ml sterile saline or B. abortus OPS vaccine/0.1ml sterile saline. The mice were challenged 5 weeks later with5×10^(5(delete4)) bacteria/0.1 ml sterile saline. They were sacrificedand assessed one week later. The results are shown in Table 1.

TABLE 1 Cross-protection study using a previous vaccine formulationGroup of mice (5 mice/group) Spleen size Average number of bacteria(CFU)/spleen Control: mice infected with B. abortus Normal 13,200 ±4,170  2308 Mice vaccinated with 1 ug OPS, infected Normal 5,960 ± 2,440with B. abortus 2308 Mice vaccinated with 100 ug OPS, infected Normal164,000 ± 95,100  with B. abortus 2308 Control: mice infected with B.suis 145 Large 4,460,000 ± 454,000   Mice vacinaed with 1 ug OPS,infected Normal 908,000 ± 719,000 with B. suis 145 Mice vaccinated with100 ug OPS, infected Normal 511,000 ± 246,000 with B. suis 145 Control:mice infected with B. melitensis Large 279,000 ± 36,800  16M Micevaccinated with 1 ug OPS, infected Normal 325,000 ± 183,000 with B.melitensis 16M Mice vaccinated with 100 ug OPS, infected Normal 102,200± 18,000  with B. melitensis 16M

By convention, minimal protection is when vaccinates have 10-fold lessbacteria in their spleens than unvaccinated controls. The above showsthat the former vaccine formulation as disclosed in U.S. Pat. No.5,951,987 was not protective against B. melitensis 16M nor B. suis 145.It was also of little effect against B. abortus 2308, an unusual strainof B. abortus that sometimes changes its antigens to evade the immunesystem of the mouse (G. G. Schurig, personal communications, 1997). Asthe previous vaccine, formulated from B. abortus 1119-3 (that expressesthe “A” OPS) was not protective against B. melitensis 16M (thatexpresses “M” OPS), B. suis 145 (that expresses “M” as well as “A” OPS)nor in this case B. abortus 2308 (which is variable and sometimes shiftsfrom “A” to “M” OPS), it was apparent that the previous vaccine (asdisclosed in U.S. Pat. No. 5,951,987) was limited in its scope ofprotection. At the time of the previous patent submission, in which thevaccine formulated from B. abortus 1119-3 was protective against thespecies and strains of Brucella tested, this was unobvious. The previouspatent and past publication taught away from the new finding that acombination vaccine, one with more than one OPS, was needed for widerprotection against Brucella.

As noted in the above table, large doses of B. abortus 1119-3 OPS (i.e.100 ug/mouse) did not offer any advantage for protection and indeedappeared to be counter-protective from B. abortus 2308 challenge. TheApplicant has observed that low doses are more effective than highdoses, one dose is better than three doses, and formulations thatenhance antibody production (e.g. OPS on lipopolysaccharide, OPS inliposomes) are counter-productive. Antibody induction iscounter-productive because antibody will opsonize, or coat, invadingbacteria, these are recognized by white blood cells which ingest thebacterium, and then the parasitic bacterium is inside the host whiteblood cell, its preferred environment. In contrast, low doses of OPSvaccine appear to stimulate cell-mediated responses (Dr. John Wyckoff,Oklahoma State University, personal communications, 2000).

EXAMPLE 2 Effectiveness of Various Combination Vaccine Candidates onVaccinated Mice Infected One Day After Vaccination

TABLE 2 Response of vaccinated mice infected one day after vaccination.Median bacterial numbers are shown in brackets. Each group representsfive mice. The animals were sacrificed seven days after infection.Vaccine given to group Spleen wt/body wt Bacterial CFU/spleen None,saline control 0.0211 ± 0.0028 2,400,000 ± 250,000   (2,200,000) 1 ug B.melitensis OPS 0.0163 ± 0.0027 1,690,000 ± 451,000   (1,860,000) 100 ugB. melitensis OPS 0.0114 ± 0.0007 850,000 ± 361,000 (880,000) 0.5 ug B.melitensis OPS + 0.5 ug B. abortus OPS 0.0172 ± 0.0012 1,820,000 ±381,000   (2,140,000) 50 ug B. melitensis OPS + 50 ug B. abortus 0.0129± 0.0015 984,000 ± 386,000 (680,000) 0.5 ug B. melitensis OPS-protein +0.5 ug B. 0.0089 ± 0.0003 624,000 ± 359,000 abortus OPS (96,000) 50 ugB. melitensis OPS-protein + 50 ug B. abortus 0.0183 ± 0.0015 1,550,000 ±351,000   OPS (1,560,000)

Throughout several years of studying Brucella infections in mice, it wasevident that not all infected mice come down with brucellosis. Usuallyabout 5-10% of the mice are infected, as evidenced by Brucella in theirspleens, but as the numbers are trivial, they are obviously notdeveloping brucellosis. In the publication by Detilleux et al.(Detilleux, P. G., Deyoe, B. L., and Cheville, N. F. 1990. Penetrationand Intracellular Growth of Brucella abortus in nonphagocytic cells invitro. Infection and Immunity 58: 2320-2328) it was observed thatBrucella takes about 2 hours to infect mammalian cells. The Applicanthas also observed over the years that the most virulent forms ofBrucella (e.g. B. melitensis 16M) shed their OPS, potentiallyvaccinating the experimental animal before infection. Two questions wereasked. Does a combination vaccine protect against more strains andserotypes of Brucella? Does this vaccine work in a very short timeframe, such as a day instead of several weeks?

In the above data, initially it appears that none of the vaccineformulations were protective when given a day before infection. That isbecause a single mouse in a group of 5 that either was not responsive tothe vaccine or if the vaccine was not properly administered, will havehigh bacterial counts that will skew the average. As the data wasentered, it was obvious that the one highlighted (0.5 ug B. melitensisOPS-protein complex+0.5 ug B. abortus OPS) was actually very protective.In this instance, the median (or the middle number) was more reflectivethan the average bacterial count. It can also be seen that the vaccinedoes protect mice against B. melitensis 16M infection, even when givenas early as a day before infection (and other studies in the Applicant'slaboratory at the Defence Research Establishment Suffield (DRES) showedvaccine protection in as little as 1 hour before infection). Thepotential is that livestock being shipped to an area of high brucellosisprevalence, or a traveller or soldier travelling to a country whereBrucella is endemic, can be vaccinated with protection developing asthey are en route.

EXAMPLE 3 Effectiveness of Various Combination Vaccine Candidates onVaccinated Mice Infected Five Weeks After Vaccination

TABLE 3 Response of vaccinated mice infected five weeks aftervaccination. Median bacterial numbers are shown in brackets. Each grouprepresents five mice. The animals were sacrificed seven days afterinfection. Vaccine given to group Spleen wt/body wt Bacterial CFU/spleenNone, saline control 0.0117 ± 0.0015 1,120,000 ± 195,000   (1,300,000) 1ug B. melitensis OPS 0.0066 ± 0.0004 48,500 ± 23,000 (44,000) 100 ug B.melitensis OPS 0.0073 ± 0.0002 365,000 ± 167,000 (290,000) 0.5 ug B.melitensis OPS + 0.5 ug B. abortus OPS 0.0061 ± 0.0004 35,800 ± 6,460 (42,000) 50 ug B. melitensis OPS + 50 ug B. abortus 0.0065 ± 0.0005162,000 ± 70,000  (150,000) 0.5 ug B. melitensis OPS-protein + 0.5 ug B.0.0059 ± 0.0003 67,600 ± 30,200 abortus OPS (92,000) 50 ug B. melitensisOPS-protein + 50 ug B. abortus 0.0069 ± 0.0004 85,100 ± 26,000 OPS(70,000)

The results shown in Tables 2 and 3 above, demonstrate that overall, 0.5ug B. melitensis OPS-protein+B. abortus OPS was most effective inproviding protection against B. melitensis 16M infection, either one dayor five weeks after vaccination. Higher dose of the same vaccine doesnot seem to be as effective, probably because it elicit antibodies.Tables 2 and 3 also show that given several weeks, OPS vaccines areeffective for protecting mice from B. melitensis 16M infections.

EXAMPLE 4 Effectiveness of Different Vaccine Candidates AgainstDifferent Species and Strains of Brucella Infections

In this case, Balb/c mice were vaccinated by the intra-peritoneal routewith various vaccine candidates. The mice were challenged (i.p.) fourweeks later with different species of Brucella. One week afterinfection, the mice were sacrificed and assessed for brucellosis. Eachgroup represents the average for five mice, each mouse was given 1 ug ofeach OPS.

TABLE 4 Effect of various vaccines against different type of Brucellainfections. Spleen wt/body wt (first line) Bacterial count (CFU) inspleen (second and third line) B. melitensis 16M B. suis 145 B. abortus30 B. abortus 2308 infection infection infection infection Control -0.0162 ± 0.0025 0.0052 ± 0.0008 0.0080 ± 0.0008 0.0047 ± 0.0004 novaccine 3,470,000 ± 740,000   346,000 ± 74,900  970,000 ± 191,000155,000 ± 74,300  B. abortus OPS 0.0077 ± 0.0012 0.0052 ± 0.0003 0.0077± 0.0014 0.0055 ± 0.0010 vaccine 270,000 ± 136,000 91,200 ± 20,500391,000 ± 146,000 7,740 ± 4,030 B. suis OPS vaccine 0.0055 ± 0.00030.0045 ± 0.0003 0.0048 ± 0.0005 0.0050 ± 0.0005 6,650 ± 2,310 20 ± 11148 ± 91  9,920 ± 5,090 B. melitensis OPS- 0.0059 ± 0.0006 0.0045 ±0.0004 0.0051 ± 0.0004 0,0065 ± 0.0008 protein 25,900 ± 13,800 0 ± 0 108± 59  238,000 ± 77,400  B. abortus OPS + B. 0.0055 ± 0.0004 0.0052 ±0.0005 0.0045 ± 0.0006 0.0053 ± 0.0005 melitensis OPS- 36,900 ± 16,40012 ± 5  212 ± 212 112,000 ± 63,300  protein B. abortus OPS + B. 0.0037 ±0.0002 0.0048 ± 0.0004 0.0050 ± 0.0003 0.0052 ± 0.0004 melitensis OPS-(no water 1 day) 1 ± 1 56 ± 51 84,900 ± 32,600 protein + B. suis OPS44,700 ± 31,400

The above results show that either a combination of OPS and OPS-proteinantigens from different Brucellae, or an OPS preparation from a singlestrain of Brucella, (for example B. suis 145) that expresses more thanone OPS, were effective in protecting mice from brucellosis. The mostremarkable of the vaccine “cocktails” tested is that of B. suis 145 OPS.It not only protects against a very wide range of Brucella species, butit also appears to work the best for each. It is believed that thisgreater OPS vaccine protection is because B. suis 145 not only expressesboth “A” and “M” (instead of just one) OPS, but that it also expressesone (which Dr. Brad Berger at DRES has isolated) or more additional andpreviously unknown OPS.

It is also interesting to note that the vaccines work best (i.e. thereis a greater contrast between non-vaccinated control mice and vaccinatedmice) for the species and strains of Brucellae that are the mostvirulent (i.e. species or strains of Brucella that cause a large spleensizes and high bacterial numbers in the spleens of unvaccinatedanimals). OPS is on the surface of smooth Brucella and it is also shedby the most virulent species and strains of Brucella. It is now believedthat the OPS is a virulence factor that reduces the resistance of thehost. By vaccinating with OPS and inducing an immunity against thiscomponent, it is likely that the host has an immunity both against theBrucella bacterium but also against the OPS that they shed.

EXAMPLE 5 Effectiveness of Different Vaccine Candidates Using VariousAdministration Routes

In this case, mice were vaccinated by different routes and variousvaccines candidates were used. The mice were challenged four weeks aftervaccination with different species of Brucella and were sacrificed andassessed one week after vaccination. Each group represents the averageof 15 mice and each mouse was given 1 ug of OPS.

TABLE 5 Effectiveness of different vaccine candidates using variousadministration of vaccine routes. Spleen wt/body wt Bacterial count(CFU) in spleen B. melitensis 16M B. suis 145 B. abortus 30 B. abortus2308 challenge, i.n. challenge, i.n. challenge, i.n. challenge, i.n.Saline control, i.m. 0.0082 ± 0.0004 0.0117 ± 0.0010 0.0047 ± 0.00010.0052 ± 0.0001 124,000 ± 34,900  448,000 ± 122,000 87 ± 24 7,040 ±2,540 B. suis OPS, i.n. 0.0088 ± 0.0004 288,000 ± 90,100  B. suis OPS,i.m. 0.0071 ± 0.0004 0.0082 ± 0.0006 0.0045 ± 0.0001 0.0048 ± 0.0001288,000 ± 90,100  48,500 ± 14,900 65 ± 27 954 ± 523 B. abortus OPS + B.0.0065 ± 0.0002 0.0067 ± 0.0005 0.0045 ± 0.0001 0.0050 ± 0.0003melitensis OPS- 22,000 ± 10,600 31,400 ± 11,600 20 ± 7  125 ± 68 protein + B. suis OPS, i.m. Saline control, s.c. 0.0074 ± 0.0004 0.01127± 0.0010  0.0049 ± 0.0001 0.0052 ± 0.0001 62,000 ± 21,900 339,000 ±94,400  230 ± 99  6,690 ± 2,040 B. suis OPS, s.c. 0.0060 ± 0.0005 0.0072± 0.0005 0.0046 ± 0.0002 0.0050 ± 0.0001 15,900 ± 12,000 29,000 ± 7,680 53 ± 19 361 ± 179 B. abortus OPS + B. 0.0064 ± 0.0002 0.0070 ± 0.00040.0047 ± 0.0001 0.0047 ± 0.0001 melitensis OPS- 7,100 ± 1,980 25,000 ±6,000  31 ± 20 154 ± 65  protein + B. suis OPS, s.c.

The above shows that either a “cocktail” vaccine, made by addingpurified antigens from different bacteria or by adding differentantigens prepared from one bacterium, given by different routes canprotect mice from a wide range of different Brucella species andstrains.

Recently the B. suis 145 OPS vaccine (1 ug/mouse) was given toanaesthetized mice (female, balb/c) by the intra-nasal route and micewere challenged 4 weeks later with B. suis 145 also given intra-nasally.Administration by this route did not appear to offer any protection. Atthe Applicant's research establishment, it has been observed thatvaccination against other infectious bacteria by the intranasal routeappears to induce antibodies in the respiratory tract (Dr. BillKournikakis, unpublished data). The induction of antibodies iscounter-productive for vaccine protection against Brucella. Recently ithas been found that B. suis 145 OPS offers protection in mice frombrucellosis when given in doses ranging from 1 ng to 100 ug. It was notprotective when doses were less than a nanogram. It is likely that thisvaccine can be effective for protecting mice from brucellosis when givenintranasally, but that the lower doses, in the nanogram rather thanmicrogram range, must be used to induce a cell-mediated response and toavoid a counter-productive antibody response.

EXAMPLE 6 Passive Immunity

Mice were vaccinated with 1 ug B. suis OPS intraperitoneally. An hourlater these were sacrificed and their serum collected. Half a ml ofserum was given to each naïve mouse about 3 hours later. Recipient micewere challenged with B. melitensis 16M an hour after receiving the notedserum. The results are shown in Table 6.

TABLE 6 Passive Immunity Group Mouse Mouse wt. (g) Spleen wt (g)Bacteria in spleen CONTROL - mice received 0.5 ml of 1 23.52 0.40345,370,000 serum from unvaccinated mice. 2 21.28 0.4682 4,280,000Infected 1 hr later, assessed 1 week 3 22.24 0.4491 2,000,000 later 420.56 0.4027 1,860,000 5 23.36 0.4618 2,580,000 TEST - mice received 0.5ml of serum 1 21.82 0.1174 620,000* from vaccinated mice. Infected 1 hr2 19.52 0.4663 260,000* later, assessed 1 week later. 3 24.66 0.21621,000,000 4 21.64 0.6244 0 5 21.52 0.6765 2,360 *colonies were unusuallysmall

The above shows that protection against brucellosis is very rapid(protection occurs within 1 hour of being vaccinated) and that thisprotection can be transferred by giving immune serum to naive mice togive them protection as well.

EXAMPLE 7 Passive Immunity (Continued)

In a second passive immunity study, mice were vaccinated with 1 ug eachwith B. melitensis 16M OPS-protein given i.p, sacrificed four weekslater, and their blood was fractionated. Naive mice (Table 7), wererecipients (the number of mice used was small for this was proof ofconcept to justify further study). A control mouse was given salineintraperitoneally. For the other groups, these received (from the firstset of sacrificed mice) their red blood cells (0.5 ml/mouse), serum (0.5ml/mouse) or washed white blood cells (in 0.3 ml saline/mouse). Micegiven these injections were challenged one day later with 5×10⁴ bacteriagiven intra-peritoneally, and were sacrificed and assessed one weeklater.

TABLE 7 Passive Immunity Mice were Body Spleen Bacteria in given: Mousewt (g) wt. (g) spleen Saline 1 37.44 0.7547 5,340,000 Red blood cells 137.52 0.4737 2,980,000 2 39.18 0.8639 2,840,000 3 37.12 0.7522 2,480,000White blood cells 1 35.52 0.4611 740,000 2 36.86 0.3960 1,860,000 336.40 0.5360 1,000,000 Serum 1 39.66 0.4098 440,000 2 35.06 0.39562,500,000 3 37.92 0.2497 280,000

The above shows that in vaccinated mice, the protective factor appearsto be made in white blood cells and then released into the serum. Thisprotective factor is produced rapidly (within 1 hour as evidenced byTable 6), continues to be produced for several weeks (as evidenced bythe above Table 7) and can be transferred to naive mice to offer themprotection from brucellosis as well.

We claim:
 1. An immunogenic composition comprising a combination ofBrucella “A” outer-polysaccharide and Brucella “M” outer-polysaccharideand Brucella “R” protein antigens, wherein said immunogenic compositionis at least free of LPS in an amount obtainable by centrifugation.
 2. Animmunogenic composition as claimed in claim 1, wherein said Bruclla “A”outer-polysaccharide and Brucella “M” outer-polysaccharide are at least90 percent pure.
 3. An immunogenic composition as claimed in claim 1,wherein said Brucella of the Brucella “A” outer-polysaccharide is aBrucella species selected from the group consisting of B. abortus biovar1, B. abortus biovar 2, B. abortus biovar 3, B. abortus biovar 6, B.melitensis biovar 2, B. suis biovar 1, B. suis biovar 2, B. suis biovar3, B. neotomae and B. maris; said “M” outer-polysaccharide is extractedfrom Brucella species selected from the group consisting of B. abortusbiovar 4, B. abortus biovar 5, B. abortus biovar 9, B. melitensis biovar1, and B. suis biovar 5; and said “R” antigens are protein, corepolysaccharide and outer-polysaccharide and Brucella of said Brucella“R” antigens is a Brucella species selected from the group consisting ofB. ovis and B. canis.
 4. An immunogenic composition as claimed in claim1, wherein said Brucella of said Brucella “A” outer-polysaccharide andsaid Brucella of said “M” outer-polyssccharide are independentlyselected from a Brucella species selected from the group consisting ofB. abortus biovar 7, B. melitensis biovar 3 and B suis biovar 4, andsaid Brucella of the Brucella “R” antigens is a Brucella speciesselected from the group consisting of B. ovis and B. canis.
 5. Animmunogenic composition comprising a combination of Brucellaouter-polysacoharides comprising Brucella “A” outer-polysaccharide,Brucella “M” outer-polysaccharide and a Brucellaouter-polysaccharide-protein complex, wherein said immunogeniccomposition is at least free of LPS in an amount obtainable bycentrifugation.
 6. An immunogenic composition as claimed in claim 5,wherein said outer-polysaccharides are at least 90 percent pure.
 7. Animmunogenic composition as claimed in claim 5, wherein said Brucella ofthe Brucella “A” outer-polysaccharlde is a Brucella species selectedfrom the group consisting of B. abortus biovar 1, B. abortus biovar 2,B. abortus biovar 3, B. abortus biovar 6, B. melitensis biovar 2, B.suis biovar 1, B. suis biovar 2, B. suis biovar 3, B. neotomae and B.maris; said Brucella of the Brucella “M” outer-polysaccharide is aBrucella species selected from the group consisting of B. abortus biovar4, B. abortus biovar 5, B. abortus biovar 9, B. melitensis biovar 1, andB suis biovar 5; and said outer-polysaccharide-protein complex isselected from the group consisting of outer-polysaccharide and Brucellamembrane proteins, outer-polysacoharide and Brucella surface proteins,outer-polysaccharide and Brucella surface enzymes andouter-polysaccharide and Brucella cytoplasmic proteins.
 8. Animmunogenic composition as claimed in claim 5, wherein said Brucella ofsaid Brucella “A” outer-polysaccharide and said Brucella of said “M”outer-polysaccharide are independently selected from a Brucella speciesselected from the group consisting of B. abortus biovar 7, B. melitensisbiovar 3 and B. suis biovar 4, and said outer-polysaccharideproteincomplex is selected from the group consisting of outer-polysaccharideand Brucella membrane proteins, outer-polysaccharide and Brucellasurface proteins, outer-polysaccharide and Brucella surface enzymes andouter-polysaccharide and Brucella cytoplasmic proteins.
 9. Animmunogenic composition as claimed in claim 1, wherein each of saidouter-polysaccharide is present in an amount equivalent to 1 ng to 100μg for mice.
 10. An immunogenic composition as claimed in claim 9,wherein said outer-polysaccharide is present in an amount equivalent to1 ug for mice.
 11. A prophylactic method of protecting againstbrucellosis comprising administering to a mammal an immunogeniccomposition as claimed in claim 1 prior to infection.
 12. A method asclaimed in claim 11, wherein said immunogenic composition isadministered intraperitonally, sub-cutaneously, intra-muscularly,intra-nasally or orally.
 13. A prophylactic method of protecting againstbrucellosis comprising administering to a mammal, serum or white bloodcells obtained from mammals administered an immunogenic composition asclaimed in claim 1.